Newcastle disease is a highly contagious' disease of poultry caused by Newcastle disease virus having an intracerebral pathogenicity index (ICPI) of 0.7 or more in chicken. In a case of unvaccinated chickens, chickens may show up to 100% mortality within 2 weeks. Chickens infected with Newcastle disease show clinical symptoms including depression, respiratory symptoms, neurologic symptoms and severe diarrhea.
Newcastle disease occurs in Asia, Africa, Europe, etc. and results in enormous economic losses in the poultry industry. In Korea, the first outbreak of Newcastle disease was recognized in the 1920s, and Newcastle disease virus was isolated for the first time in 1949. Newcastle disease has not yet been eradicated in Korea and the economic impact is enormous. In areas including Asia (including Korea) and Africa in which Newcastle disease is enzootic, vaccination policy has been implemented for protection of chickens against Newcastle disease. Currently, Newcastle disease vaccines are formulated using Newcastle disease virus having an intracerebral pathogenicity index of less than 0.4 (hereinafter referred to non-pathogenic virus) in the form of either live vaccines or inactivated killed vaccines.
Taxonomically, Newcastle virus is an RNA virus belonging to the family Paramyxoviridae, and the viral genome of an about 15.2 K nucleotide encodes at least 6 structural proteins, namely NP, P, M, F, HN and L. Among them, the F protein is a structural protein constituting the envelope of Newcastle disease virus along with the HN protein and induces a virus-neutralizing antibody in a host and plays an important role in the defense of a host against disease.
The mechanism of virus replication in susceptible host cells is as follows. The HN protein of the envelope of Newcastle disease virus attaches to the host cell membrane, and then the fusion between the viral envelope and the host cell membrane occurs, while a genome-containing nucleocapsid in the virions is introduced into the host cells. Then, the viral genome replicates genomic RNA using the metabolic system of the host cells and produces viral structural proteins, and thus the proteins are assembled into virion in the cells. Then, the viral particles are released from the cells via a budding process to produce infectious progeny viruses. Herein, the F protein of Newcastle virus is involved in the fusion process between the viral envelope and the cytoplasm membrane to promote virus infectivity in the cell. Specifically, the F protein is present in the form of an inactive precursor (F0) in the virion. When the virion attaches to the host cells using viral HN protein, the inactive precursor F0 is cleaved into active subunits F1 and F2 by the protease of the host cells. The subunits F1 and F2 thus lead to membrane fusion between virion and cell.
It is known that the virulence of Newcastle disease virus depends on how easily the inactive precursor F0 is cleaved into the active subunits F1 and F2 in susceptible host cells. Pathogenic Newcastle disease virus having a polybasic amino acid sequence (-Arg/Lys-Arg-Gln-Lys/Arg-Arg-Phe-) (SEQ ID NO: 12) at the F1/F2 cleavage site can be easily cleaved by ubiquitous proteases in a wide range of host cell types, whereas non-pathogenic Newcastle disease virus has no polybasic amino acid sequence, and for this reason, the cleavage sites can be cleaved only by trypsin-like enzymes, which are found in limited areas such as respiratory mucosal tissue.
Tentative diagnosis of Newcastle disease can be determined based on the clinical signs of affected birds and epidemiological history. Definite diagnosis should be achieved by laboratory tests. Laboratory tests for diagnosis of Newcastle disease generally include methods for detection of Newcastle disease virus antigen and/or antibody that are specific for Newcastle disease virus.
The antigen detection methods using a few tissue samples containing high titered viral load from diseased chickens or dead chickens are effective in diagnosing Newcastle disease. Currently available antigen detection methods include virus isolation, reverse transcriptase-polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA).
The antibody detection methods can be used to detect Newcastle disease virus specific antibodies in unvaccinated poultry or to measure significantly increased antibody titer in vaccinated poultry after onset of outbreak. Currently available antibody detection methods include hemagglutination inhibition (HI) test, virus neutralization (VN) test, and enzyme-linked immunosorbent assay (ELISA). The antibody detection methods are very effective in testing large-scale samples, because they can be performed in living chickens and can test a large amount of samples within a short time compared to the antigen detection methods. However, currently available antibody detection methods have a disadvantage in that they cannot differentiate infected individuals from vaccinated individuals.
In most of the areas or countries with disease outbreaks or at risk, vaccination is performed in order to protect susceptible chickens from Newcastle disease. In these areas, some vaccinated but unsolid immunized chickens can be infected with pathogenic Newcastle virus. Most of such chickens show subclinical symptoms without any death, but can shed a small amount of pathogenic Newcastle virus through feces or respiratory organs. Chickens carrying Newcastle disease virus thus can be considered as a reservoir that may play an important role in spreading disease. This is because, on farms in which several tens of thousands of chickens are raised, it is difficult to detect some virus-harbouring chickens in a chicken flock by clinical inspection alone. Currently the most reliable method is to use specific pathogen-free (SPF) chickens as sentinel birds in order to detect pathogenic Newcastle disease virus circulating in the chicken flock. However, this method is not very suitable, because the purchase and management of the sentinel chickens are very troublesome and, in addition, the sentinel chickens can act as a source of spreading contagious diseases. The antigen detection methods can be a method to detect infected chickens among uninfected chickens, but it is also not suitable, because the titer of virus shed from vaccinated and infected chickens is low and these methods are not practical to test a large number of chickens at the same time.
The most effective method of detecting infected chickens among a large-scale chicken flock at the same time is to use a serological test method that can differentiate infected from vaccinated animals (DIVA). Currently available serological test methods include a hemagglutination inhibition (HI) test method and an enzyme-linked immunosorbent assay. However, these methods cannot differentiate infected animals from vaccinated animals. Therefore, plausible strategies for realizing the differentiation between infected and vaccinated animals (DIVA) are generally classified into two categories. The first method is a method comprising vaccination in chicken with a marker vaccine with lack of non-protective Newcastle disease viral protein, and examining the presence of antibodies to the deleted protein in birds. This strategy requires obligatory use of the marker vaccine instead of the current whole virus. The second method employs a diagnostic method for detecting an antibody to an antigenic site (epitope) that is present only in pathogenic Newcastle disease virus. However, this diagnostic method is not yet developed.